Recombinant fusion proteins and libraries from immune cell repertoires

Abstract

Disclosed herein are methods and compositions for generating a repertoire of recombinant fusion polypeptides from immune cells, and uses thereof.

The invention claimed is: 
     
       1. A kit for producing a library of constructs for expression of thousands of full-length recombinant immunoglobulins, the kit comprising:
 a. a plurality of at least 10,000 unique recombinant fusion polynucleotides, each comprising a first polynucleotide and a second polynucleotide and a linker polynucleotide,
 each first polynucleotide encoding a heavy chain variable domain from a cognate pair from a single isolated mammalian B cell and an IgG heavy chain constant domain fragment; and 
 each second polynucleotide encoding a light chain variable domain from the cognate pair from the single isolated mammalian B cell and an IgK light chain constant domain; and 
 the linker polynucleotide linking the first and second polynucleotides and comprising a restriction site; 
 
 b. a third recombinant polynucleotide, not operationally linked to the recombinant fusion polynucleotides, comprising a first promoter sequence and a sequence encoding a portion of the IgG heavy chain constant domain, wherein the third recombinant polynucleotide comprises a first end sequence and a second end sequence, each overlapping with a first or second end sequence of each recombinant fusion polynucleotide; 
 c. a fourth recombinant polynucleotide, not operationally linked to the recombinant fusion polynucleotides or the third recombinant polynucleotide, comprising a second promoter sequence or a translational skip site; and 
 d. a mixture for Gibson assembly. 
 
     
     
       2. The kit of  claim 1 , wherein each single isolated mammalian cell is isolated from a human donor immunized with an antigen. 
     
     
       3. The kit of  claim 1 , comprising at least 100,000 unique recombinant fusion polynucleotides. 
     
     
       4. The kit of  claim 1 , wherein the first promoter sequence is an AOX1 promoter sequence. 
     
     
       5. The kit of  claim 1 , wherein the fourth recombinant polynucleotide comprises the translational skip site. 
     
     
       6. The kit of  claim 4 , wherein the fourth recombinant polynucleotide comprises the translational skip site. 
     
     
       7. The kit of  claim 1 , wherein the fourth recombinant polynucleotide comprises the second promoter sequence. 
     
     
       8. The kit of  claim 4 , wherein the fourth recombinant polynucleotide comprises the second promoter sequence. 
     
     
       9. The kit of  claim 1 , wherein the fourth recombinant polynucleotide comprises the second promoter sequence and the second promoter sequence is an AOX1 promoter. 
     
     
       10. The kit of  claim 4 , wherein the fourth recombinant polynucleotide comprises the second promoter sequence and the second promoter sequence is an AOX1 promoter.